In the last decade there has been an explosive growth in the field of ubiquitin research, with approximately 530 human genes predicted to encode enzymes involved in the conjugation and deconjugation of ubiquitin. Of these 95 encode deubiquitylases (DUBs) and these enzymes have become the target of many on-going drug discovery efforts. All DUBs carry-out the enzymatic removal of ubiquitin (Ub) from target proteins, whether that protein is a normal cellular protein or is Ub itself. Deconjugation requires the cleavage of an isopeptide bond formed between the C-terminus of Ub and the -NH2-group of a Lys residue in the target protein. With the exception of the JAMM-family of DUBs, all DUBs fall into the peptidase C19 family and have an activated cysteine residue in the active site. As a consequence, most screening programs have a high hit rate of cys-reactive compounds that show low selectivity and potency. The most potent DUB inhibitors are generally produced by incorporation of a cys-reactivity moiety at the C-terminus of Ub, e.g. Ub-aldehyde, Ub-vinylsulfone, Ub-vinylmethylester. By their very nature, i.e. relying on the binding of Ub to the enzyme, such inhibitors exhibit little or no selectivity between different DUBs. The field of Ub research and the validation of DUBs as targets for drug discovery efforts, would be dramatically advanced by the availability of selective, high potency inhibitors of specific DUBs. In this application, we propose to develop panels of such inhibitors. In order to achieve high potency, we will take advantage of the reactivity of the active site cysteine through the use of a -lactam scaffold. Attack on the lactam ring by the active site Cys leads to the generation of a stable tetrahedral intermediate. Selectivity is obtained by the substituents used to decorate the ring. This approach has been used by others to produce highly selective inhibitors of a number of enzymes, e.g. the blood coagulation enzymes activated factor X (FXa) and activated factor XI (FXIa), leukocyte elastase, tryptase, and cathepsins. It should be noted that the cathepsins are cysteine proteases, demonstrating that lactam-based inhibitors will function against this mechanistic class. Although our primary goal in this project is to generate a new series of research tools, the compounds that we produce could be used as lead compounds for drug development. Finally, the SAR that we develop around these compounds can be used to guide Medicinal Chemistry efforts around other compounds.